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A Simple, Improved Method for Scarless Genome Editing of Budding Yeast Using CRISPR-Cas9
Until recently, the favored method for making directed modifications to the budding yeast genome involved the introduction of a DNA template carrying the desired genetic changes along with a selectable marker, flanked by homology arms. This approach both limited the ability to make changes within ge...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9607540/ https://www.ncbi.nlm.nih.gov/pubmed/36287051 http://dx.doi.org/10.3390/mps5050079 |
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author | Aguilar, Rhiannon R. Shen, Zih-Jie Tyler, Jessica K. |
author_facet | Aguilar, Rhiannon R. Shen, Zih-Jie Tyler, Jessica K. |
author_sort | Aguilar, Rhiannon R. |
collection | PubMed |
description | Until recently, the favored method for making directed modifications to the budding yeast genome involved the introduction of a DNA template carrying the desired genetic changes along with a selectable marker, flanked by homology arms. This approach both limited the ability to make changes within genes due to disruption by the introduced selectable marker and prevented the use of that selectable marker for subsequent genomic manipulations. Following the discovery of CRISPR-Cas9-mediated genome editing, protocols were developed for modifying any DNA region of interest in a similar single transformation step without the need for a permanent selectable marker. This approach involves the generation of a DNA double-strand break (DSB) at the desired genomic location by the Cas9 nuclease, expressed on a plasmid which also expresses the guide RNA (gRNA) sequence directing the location of the DSB. The DSB is subsequently repaired via homologous recombination using a PCR-derived DNA repair template. Here, we describe in detail an improved method for incorporation of the gRNA-encoding DNA sequences into the Cas9 expression plasmid. Using Golden Gate cloning, annealed oligonucleotides bearing unique single-strand DNA overhangs are ligated into directional restriction enzyme sites. We describe the use of this CRISPR-Cas9 genome editing protocol to introduce multiple types of directed genetic changes into the yeast genome. |
format | Online Article Text |
id | pubmed-9607540 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-96075402022-10-28 A Simple, Improved Method for Scarless Genome Editing of Budding Yeast Using CRISPR-Cas9 Aguilar, Rhiannon R. Shen, Zih-Jie Tyler, Jessica K. Methods Protoc Protocol Until recently, the favored method for making directed modifications to the budding yeast genome involved the introduction of a DNA template carrying the desired genetic changes along with a selectable marker, flanked by homology arms. This approach both limited the ability to make changes within genes due to disruption by the introduced selectable marker and prevented the use of that selectable marker for subsequent genomic manipulations. Following the discovery of CRISPR-Cas9-mediated genome editing, protocols were developed for modifying any DNA region of interest in a similar single transformation step without the need for a permanent selectable marker. This approach involves the generation of a DNA double-strand break (DSB) at the desired genomic location by the Cas9 nuclease, expressed on a plasmid which also expresses the guide RNA (gRNA) sequence directing the location of the DSB. The DSB is subsequently repaired via homologous recombination using a PCR-derived DNA repair template. Here, we describe in detail an improved method for incorporation of the gRNA-encoding DNA sequences into the Cas9 expression plasmid. Using Golden Gate cloning, annealed oligonucleotides bearing unique single-strand DNA overhangs are ligated into directional restriction enzyme sites. We describe the use of this CRISPR-Cas9 genome editing protocol to introduce multiple types of directed genetic changes into the yeast genome. MDPI 2022-10-04 /pmc/articles/PMC9607540/ /pubmed/36287051 http://dx.doi.org/10.3390/mps5050079 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Protocol Aguilar, Rhiannon R. Shen, Zih-Jie Tyler, Jessica K. A Simple, Improved Method for Scarless Genome Editing of Budding Yeast Using CRISPR-Cas9 |
title | A Simple, Improved Method for Scarless Genome Editing of Budding Yeast Using CRISPR-Cas9 |
title_full | A Simple, Improved Method for Scarless Genome Editing of Budding Yeast Using CRISPR-Cas9 |
title_fullStr | A Simple, Improved Method for Scarless Genome Editing of Budding Yeast Using CRISPR-Cas9 |
title_full_unstemmed | A Simple, Improved Method for Scarless Genome Editing of Budding Yeast Using CRISPR-Cas9 |
title_short | A Simple, Improved Method for Scarless Genome Editing of Budding Yeast Using CRISPR-Cas9 |
title_sort | simple, improved method for scarless genome editing of budding yeast using crispr-cas9 |
topic | Protocol |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9607540/ https://www.ncbi.nlm.nih.gov/pubmed/36287051 http://dx.doi.org/10.3390/mps5050079 |
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